Tracing the Holocene hybrid origin of cultivated walnut in southwestern China

Genetic exchanges underpin population adaptation and species evolution. However, knowledge of the genetic impacts of historical, anthropogenic crop-to-wild gene flow remains limited. In southwestern China, the long cultivation and parapatric distribution of Juglans regia and J. sigillata provide an ideal system to investigate this process, yet the evolutionary origin of the cultivated walnut in this region remains a mystery. Using 31 microsatellite loci to genotype 2,866 individuals, along with 716 chloroplast genomes, we evaluated genetic diversity, population structure, and maternal lineage patterns across the region. Population demographic histories were subsequently inferred using approximate Bayesian computation. Our analysis revealed that J. sigillata has higher genetic diversity than J. regia, attributed to its long-term persistence in heterogeneous environments. We observed extensive hybridization between the two species around the Sichuan Basin, forming two geographically distinct subgroups: Hybrid1 in the northwest and Hybrid2 in the south. Both subgroups exhibit variable parental contributions, but with the chloroplast genome entirely introgressed from J. regia. Furthermore, the Sichuan Basin and Yangtze River acted as major geographic barriers to north-south gene flow. Our divergence time estimates showed that the two species diverged during the Pleistocene, followed by an admixture event between J. regia and J. sigillata, forming Hybrid2 during the early Holocene. Subsequent admixture between Hybrid2 and J. regia produced Hybrid1 during the middle Holocene. We propose that cultivated walnuts in southwestern China originated through introgression between J. regia and wild J. sigillata during the Holocene, prior to the introduction of agriculture to the region, a process likely facilitated by historical southward human migrations. This research elucidates how long-term human influence has subtly reshaped the genetic landscape of walnuts in southwestern China through gene flow, providing valuable insights for the study and management of other tree crops.